Cryptococcus neoformans is a major pathogen in immunocompetant as well as immunocompromised patients including those with AIDS in both the developed as well as the developing world. Our long-term objective is to test the hypothesis that molecular regulators of the virulence factor laccase affect the virulence of Cryptococcus neoformans. The specific hypothesis behind the present proposal is that a virulence associated DEAD-box protein, Vad1 and a high affinity copper transporter, Ctr4 plays a role in the virulence of the fungus. In 2013, we completed our work demonstrating a role for Vad1-dependent modulation of the immune response in pulmonary cryptococcosis and a role for Ctr4 in tolerance to starvation and brain dissemination. Vad1 is an important regulator of mRNA stability in the fungus and had previously been shown to regulate the Mata pheromone involved in mating. During the 2013 period, we have demonstrated a role for Vad1 in the regulation of autophagy by its role in modulating mRNA stability of several autophagy-dependent transcripts. Overexpression of Vad1 suppresses the transcripts resulting in suppression of autophagy induction and reduced fungal virulence. In addition to laccase activity, copper is required for essential cellular processes such as respiration, iron uptake and at least two virulence factors, the Cu, Zn superoxide dismutase and urease. This also suggests that a successful host strategy might be to sequester this important metal away from the pathogen, making the fungus compete with the mammalian host for available copper. To address this issue we used a copper dependent cuf1 strain defective in a Cu dependent regulatory factor, CUF1. The strain was shown to demonstrate reduced virulence using both an intravenous and an intranasal mouse model and expression levels of one of its target genes, a high affinity copper transporter, Ctr4 was found to correlate with dissemination to brains of patients (vs. lung disease only) in a cohort of organ transplant patients. Since the last progress report, we sought to more directly assess the role of copper homeostasis by using direct analytical copper measurements during pathogenesis and studying the role of the Ctr4 high affinity copper transporter. In a collaboration with T. OHalloran at the Univ. Chicago, we used inductively-coupled mass spectroscopy (ICP-MS) to measure intracellular copper concentrations of wt C. neoformans as well as the Cuf1 hypovirulent strain under a number of different conditions as well as after isolation of pure fungal cells from infected mouse brains by a method developed in our laboratory. Interestingly, we found that C. neoformans acquired up to three times the intracellular quota of copper compared to non-pathogenic yeasts such as S. cerevisiae and that this was maintained during infection of brains in mice. In contrast, the cuf1 mutant acquired lower levels of copper than wild-type cells, consistent with the Cuf1 role in copper homeostasis. In a second set of studies, a ctr4 mutant strain was constructed which identified a role for the copper transporter in virulence in mice, helping to support the clinical expression studies above and provided support for the role of a copper transporter in microbial pathogenesis ( highlighted by JD Shrout in Science Translational Medicine.